In the present specification, an unsaturated fatty acid refers to a fatty acid having one or more double bond(s) in a carbon chain and, among this, the fatty acid having a carbon number of 18 or more and two or more double bonds is generally referred to as “highly unsaturated fatty acid”. Examples of the highly unsaturated fatty acid include γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, mead acid, 6,9-octadecadienoic acid, 8,11-eicosadienoic acid, etc. Most of the highly unsaturated fatty acids generated by a Mortierella microorganism are produced as fatty acids constituting triglyceride and, besides this, also as fatty acids constituting various lipids such as diglyceride, monoglyceride, free fatty acid, phospholipid, etc. As used herein, the highly unsaturated acid-containing lipid is a mixture of lipids containing these various highly unsaturated fatty acids, and an amount of arachidonic acid production is a value obtained by converting the amount of arachidonic acid existing as fatty acids constituting those various lipids into the amount of free fatty acids. In addition, a mixture of oligoglucose and glucose, which is obtained by treating a starch with a saccharifying enzyme such as amylase, is referred to as “saccharified starch”, a sugar degrading enzyme such as amylase which is used for producing a saccharified starch is referred to as “saccharifying enzyme”, and treatment of a starch with the enzyme is referred to as “saccharification”. A degradation degree of starch by saccharification is referred to as “saccharification degree”, and the saccharification degree is expressed by the ratio of reducing sugar/total sugar (%) of the saccharified starch. For example, a ratio of reducing sugar/total sugar of 50% indicates that the average chain length of α-glucan in the saccharified starch is 2.
Highly unsaturated fatty acids such as arachidonic acid, along with DHA (docosahexaenoic acid), have drawn attention from a nutritional point of view, especially as a component necessary for the growth of a baby. Lanting et al. conducted a 9-year follow-up study of babies who had been breastfed and babies who had been powdered formula-fed for three weeks or longer after birth. They examined the incidence of a minor neurological dysfunction from behavioral aspects and, as a result, reported that the incidence of neurological dysfunction in children who were powdered formula-fed was 2-fold that of children who were breastfed (LANCET, vol.344, 1319-1322 (1994)). According to the report, the result is due to the fact that highly unsaturated fatty acids such as DHA, arachidonic acid, etc., which are present in breast milk but are hardly present in powdered formula, are involved in the development of a baby's brain. Besides, results that highly unsaturated fatty acids are involved in the development of brain and retina of a neonate, have been frequently reported (Carlson et al., Broc. Natl. Acad. Sci. 90:1073-1077(1993)), and the importance of those highly unsaturated fatty acids is drawing attention from a nutritional viewpoint for a premature baby and a neonate.
Highly unsaturated fatty acids are widely distributed in the living world and, for example, arachidonic acid has been separated from a lipid extracted from animal adrenal gland or liver. However, since a content of the highly unsaturated fatty acid in animal organs is small, the extraction and separation of highly unsaturated fatty acids from an animal organ is not a sufficient method for supplying a large amount of highly unsaturated fatty acids. For this reason, methods of obtaining highly unsaturated fatty acids by culturing various microorganisms have been developed. Among the microorganisms, a Mortierella microorganism is known as a microorganism producing a highly unsaturated fatty acid-containing lipid such as arachidonic acid, dihomo-y-linolenic acid, eicosapentaenoic acid, etc., and a process for producing a highly unsaturated fatty acid-containing lipid by fermentation using this microorganism has been developed (JP-A No. 63-44891, JP-A No. 63-12290, JP-A No. 63-14696, JP-A No. 63-14697). Further, a process for producing a mead acid using a mutant in which Δ12 desaturation enzyme activities of a Mortierella microorganism are reduced or lost has also been known (JP-A No. 5-91888). In addition, a process for producing dihomo-γ-linolenic acid using a mutant in which Δ5 desaturation enzyme activities are reduced or lost, which is obtained by subjecting a Mortierella microorganism to mutation induction, has also been known (JP-A No. 5-91887).
Although it has already been known that a Mortierella microorganism has the ability to assimilate a starch, since its starch-assimilating ability is inferior as compared to the glucose-utilizing ability, glucose has been widely used as a medium carbon source for such microorganism( Shinmenet al., Appl. Microbiol. Biotechnol. 31:11-16 (1989), Aki et al., JAOCS 78:599-604 (2001)). As a result of paying attention to the starch-utilizing ability of a Mortierella microorganism, it has been reported that a secreted starch-degrading enzyme is isolated and purified, and the starch-degrading enzyme is identified to be α-glucosidase (Tanaka et al., Bulletin of Japan Society of Bioscience, Biotechnology, and Agrochemistry, March 1999). However, α-glucosidase, which is an exo-type amylase having a degradation pattern of cleavage of glucose units from a non-reducing end of α-glucan, has a low activity to degrade high-molecular α-glucan, a representative of which is a starch, and has a high activity of degrading an oligoglucose composed of several glucoses. This is thought to be one of the reasons why the starch-utilizing ability of a Mortierella microorganism is lower than the glucose-utilizing ability. On the other hand, when glucose is used as a carbon source, since an increase in osmotic pressure due to high concentration of glucose in the culture medium has adverse influence on the growth of cells of, and the productivity of a highly unsaturated fatty acid-containing lipid of a Mortierella microorganism, there has been widely used a method comprising culturing the microorganism with low concentration glucose and sequentially adding glucose during culture to compensate the utilized glucose (Shinmen et al., Appl. Microbiol. Biotechnol. 31:11-16(1989)). In addition, an attempt to produce a highly unsaturated fatty acid-containing lipid using a Mortierella microorganism having resistance to high concentration glucose (International Publication No. WO98/39468) has also been tried. A carbon source of the culture medium accounts for the majority of raw material costs and, if this can be changed to a raw material which is less expensive than glucose, the cost for producing a highly unsaturated fatty acid-containing lipid can be reduced. When a starch which is a raw material for glucose is used as a medium carbon source, the raw material cost can be reduced, however, α-glucosidase produced by a Mortierella microorganism has the low activity of degrading a starch as described above and, therefore, the starch can not be sufficiently assimilated by said microorganism. In addition, a culture medium containing glucose at a high concentration has a high osmotic pressure, and this becomes a cause for delay of the growth of a Mortierella microorganism, reduction in its productivity of a highly unsaturated fatty acid-containing lipid and change in the morphological form. Therefore, it is difficult to produce a highly unsaturated fatty acid-containing lipid at a low cost by a batch culture method in which glucose is not sequentially added during culturing and glucose is contained at a high concentration at the time of the initiation of culturing. As a means to solve this problem, a feeding culture method in which glucose is sequentially added to the culture medium is used. However, since the feeding culture not only requires an additional apparatus for sequentially adding glucose but also increases the number of works and working time in the culturing step, this method increases the cost for producing a highly unsaturated fatty acid-containing lipid. In addition, although glucose is produced by enzymatic degradation of starch with various amylases, it is necessary to isolate and purify glucose after the enzymatic treatment. Thus, the cost for enzymatic treatment and isolation and purification makes glucose more expensive than starch.
Production of a highly unsaturated fatty acid-containing lipid utilizing a starch or a soluble starch as a medium carbon source has been reported (Shinmen et al., Appl. Microbiol. Biotechnol. 31:11-16(1989), Aki et al., JAOCS 78:599-604 (2001)). However, the yield of a highly unsaturated fatty acid-containing lipid per sugar in said culture medium is lower than that of the case where the same amount of glucose is used as a carbon source due to the incomplete assimilation of a starch or a soluble starch.